Synergistic actions by multiple Toll-like receptors in alcoholic liver disease

描述（由申请人提供）：慢性酒精滥用是美国成年患者肝硬化和肝功能衰竭的主要原因。酒精性肝病患者可从脂肪变性发展为脂肪性肝炎、纤维化和肝硬化。目前的概念是，慢性酒精摄入增加肠通透性，导致门静脉内毒素（脂多糖，LPS）水平升高。门静脉内毒素增加，通过toll样受体（TLR） 4刺激库普弗细胞，TLR 4是LPS的受体，可促进肝脏炎症，导致酒精性肝损伤。酒精性肝硬化患者不仅LPS升高，而且血液和腹水中的细菌DNA水平也升高。细菌DNA被TLR9识别，TLR9在免疫细胞中广泛表达，包括肝内的巨噬细胞Kupffer细胞。另一方面，我们最近发现TLR4在肝纤维化中直接激活肝星状细胞（hsc）。我们假设过量饮酒破坏肠上皮屏障导致肠道菌群来源的LPS和细菌DNA易位进入肝脏。LPS和细菌DNA分别激活Kupffer细胞和hsc中表达的TLR4和TLR9，进而产生炎症和纤维化介质，导致酒精性脂肪变性、脂肪性肝炎（ASH）和纤维化。TLR4和TLR9之间的协同相互作用以及肝细胞对细胞死亡的敏感性增加可能进一步加剧ASH和纤维化的程度。经乙醇处理后，肝脏中的Kupffer细胞产生炎性细胞因子，这可能与包括脑损伤在内的全身器官损伤有关。基于这些假设，本提案的目的是：目的1：确定TLR4在ASH中Kupffer细胞和hsc活化中的作用；产生tlr4 -骨髓嵌合体，用灌胃乙醇饲养处理。评估脑损伤和肠通透性。目的2：确定TLR9在ASH中的作用；肝脏中负责TLR9的细胞类型将在ASH模型中进行评估。目的3：确定TLR4和TLR9在库普弗细胞和造血干细胞中的协同作用。目的4：确定ASH患者肝细胞对细胞死亡的敏感性是否增加。我们将使用连续灌胃乙醇喂养模型来测试这些具体目标。本研究将深入了解多种TLR信号在酒精诱导的肠-肝-脑相互作用中的分子机制。

DESCRIPTION (provided by applicant): Chronic alcohol abuse is the major cause of cirrhosis and liver failure in adult patients in the United States. Alcoholic liver disease in patients progresses from steatosis to steatohepatitis, fibrosis, and cirrhosis. The current concept is that chronic alcoholic intake increases intestinal permeability that leads to an elevation of endotoxin (lipopolysaccharide, LPS) levels in the portal vein. Increased endotoxin via the portal vein stimulates Kupffer cells through Toll-like receptor (TLR) 4, a receptor for LPS, which promotes hepatic inflammation resulting in alcoholic liver injury. Not only LPS, but also bacterial DNA levels in blood and ascites are elevated in patients with alcoholic-induced liver cirrhosis. Bacterial DNA is recognized by TLR9 that is widely expressed in immune cells including Kupffer cells, resident macrophages in the liver. On the other hand, we have recently shown that TLR4 directly activates hepatic stellate cells (HSCs) in hepatic fibrosis. We hypothesize that excessive alcohol intake disrupts intestinal epithelial barrier leads to translocation of intestinal microflora- derived LPS and bacterial DNA into the liver. LPS and bacterial DNA activate TLR4 and TLR9, respectively, expressed in Kupffer cells and HSCs, which in turn produce inflammatory and fibrogenic mediators, resulting in alcoholic steatosis, steatohepatitis (ASH) and fibrosis. Synergistic interaction between TLR4 and TLR9, and increased sensitivity of hepatocytes to cell death might further exacerbate the degrees of ASH and fibrosis. Upon ethanol treatment, Kupffer cells in the liver produce inflammatory cytokines, which could be associated with systemic organ injury including brain injury. Based on these hypotheses, the aims of this proposal are: Aim #1: To determine the role of TLR4 on the activation of Kupffer cells and HSCs in ASH; TLR4-bone marrow chimera will be generated and treated with intragastric ethanol feeding. Brain injury and intestinal permeability will be assessed. Aim #2: To determine the role of TLR9 in ASH; The responsible cell types for TLR9 in the liver will be assessed in ASH models. Aim #3: To determine the synergistic actions by TLR4 and TLR9 in Kupffer cells and HSCs. Aim #4: To determine whether the sensitivity of hepatocytes to cell death is increased in ASH. We will test these specific aims using the continuous intragastric ethanol feeding model. The proposed study will provide insight into the molecular mechanism underlying the role of multiple TLR signaling in Gut- Liver-Brain interaction in alcohol-induced pathogenesis. PUBLIC HEALTH RELEVANCE: Alcoholic steatohepatitis represents a massive health care burden worldwide. The goal of this study is to investigate whether TLR4 and TLR9 synergistically promote alcoholic steatohepatitis and subsequent brain injury. The results from this study will provide the potential targeting for the therapy of alcohol-induced organ pathogenesis including steatohepatitis and brain injury.